Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations

ABSTRACT

Enriched neural stem and progenitor cell populations, and methods for identifying, isolating and enriching for neural stem cells using reagent that bind to cell surface markers, are provided.

CLAIM OF PRIORITY

This application is a 35 U.S.C. §371 entry of PCT/US00/03592 filed Feb.11, 2000, which claims priority to U.S. provisional patent application60/119,725, filed Feb. 12, 1999; and is a continuation-in-part of UnitedStates utility patent application Ser. No. 09/422,844, filed Oct. 21,1999, now U.S. Pat. No. 6,468,794 which claims priority to U.S.provisional patent application 60/168,407, filed Dec. 1, 1999.

TECHNICAL FIELD

This invention relates generally to enriched neural stem cell andprogenitor cell populations, and methods for identifying, isolating andenriching for neural stem and progenitor cells, particularly centralnervous system neural stem cells and progenitor cells, and mostparticularly to enriched populations of neurosphere initiating cells(NS-IC).

BACKGROUND OF THE INVENTION

Stem cell populations constitute only a small percentage of the totalnumber of cells, but are of immense interest because of their ability torepopulate the body. The longevity of stem cells and the disseminationof stem cell progeny are desirable characteristics. There is significantcommercial interest in these methods because stem cells have a number ofclinical uses. There is also medical interest in the use of stem cellsas a vehicle for gene therapy.

Proteins and other cell surface markers found on stem cell andprogenitor cell populations are useful in preparing reagents for theseparation and isolation of these populations. Cell surface markers arealso useful in the further characterization of these important cells.

Yin et al., U.S. Pat. No. 5,843,633, incorporated herein by reference,describes a monoclonal antibody called AC133, which binds to a surfacemarker glycoprotein on hematopoietic stem and progenitor cells. TheAC133 antigen is a 5-transmembrane cell surface antigen with a molecularweight of 117 kDa. Expression of this antigen is highly tissue specific,and has been detected on a subset of hematopoietic progenitor cellsderived from human bone marrow, fetal bone marrow and liver, cord blood,and adult peripheral blood. The subset of cells recognized by the AC133antibody is CD34^(bright), and contains substantially all of the CFU-GMactivity present in the CD34⁺ population, making AC133 useful as areagent for isolating and characterizing human hematopoietic progenitorand stem cells.

However, surface markers specific to non-hematopoietic stem cells andprogenitor cells, and particularly central nervous system neural stemcells and progenitor cells have not been identified. Further, the AC133antibody has not been used in methods for identifying, isolating, orenriching for non-hematopoietic stem cells or progenitor cells,particularly central nervous system (CNS) neural stem cells andprogenitor cells. There remains a need for tools, such as monoclonalantibodies that are useful in isolating and characterizing humannon-hematopoietic progenitor and stem cells, and particularly centralnervous system (CNS) neural stem cells and progenitor cells.

SUMMARY OF THE INVENTION

This invention provides methods for identifying, isolating, andenriching for human non-hematopoietic progenitor and stem cells, andparticularly central nervous system (CNS) neural stem cells which caninitiate neurospheres (NS-IC) and progenitor cells. The invention alsoprovides for enriched populations containing CNS neural stem cells thatcan initiate neurospheres, and progenitor cells. A “neurosphereinitiating cell (NS-IC)” is a cell that can initiate long-termneurosphere culture. A “neurosphere”, in turn, is an aggregate orcluster of cells which includes neural stem cells and primitiveprogenitors. The identification, culture, growth, and use ofneurospheres is disclosed in Weiss et al., U.S. Pat. No. 5,750,376 andWeiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. While the term “NS-IC” is defined by the ability or capacityof that cell to form a neurosphere, these cells may be appropriatelygrown in adherent culture (see, for example, Johe, U.S. Pat. No.5,753,506, incorporated herein by reference), and it should be notedthat the methods and populations described herein are not to be limitedto suspension cultures of NS-IC. An NS-IC is nestin+ and has thecapability to differentiate, under appropriate differentiatingconditions, to neurons, astrocytes, and oligodendrocytes.

According to one embodiment of this invention, enriched populations ofnon-hematopoietic stem cells and progenitor cells, preferably CNS neuralstem cells including NS-ICs, and progenitor cells, and method ofidentifying, isolating, or enriching for such cells, is achieved bycontacting a population of cells containing at least one stem cell orNS-IC, or progenitor cell with a reagent that binds to surface markerglycoprotein antigen (“AC133 antigen”) recognized by the AC133 antibody.In a preferred embodiment the reagent is the AC133 antibody (the AC133antibody is alternately referred to herein as “5F3”). Use of traditionaltechniques for cell sorting, such as by immunoselection (e.g., FACS),then permits identification, isolation, and/or enrichment for cells inwhich contact between the reagent and the AC133 antigen has beendetected.

In another embodiment, this invention provides a novel antibody, hereincalled 5E12, that may be used to provide enriched populations ofnon-hematopoietic stem cells and progenitor cells, preferably CNS neuralstem cells that can initiate neurospheres and progenitor cells, and maybe used in methods of identifying, isolating, or enriching for suchcells, by contacting a population of cells containing at least one stemcell NS-IC, or progenitor cell with the 5E 12 antibody, which binds to asurface marker glycoprotein antigen other than the AC133 antigen.

In a preferred embodiment, the cells of this invention, preferably theCNS neural stem cells, are additionally characterized as lacking cellsurface markers for CD45 and CD34.

In a further embodiment, this invention provides a novel antibody,herein called 8G1, believed to recognize CD24, which permitssubselection between populations of CNS neural stem cells (characterizedas 8G1^(−/lo)) and populations of CNS progenitor cells (characterized as8G1⁺).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram illustrating the proliferation and differentiationof a NS-IC.

FIG. 2 is a series of photographs showing that neurosphere cultures canbe initiated from single-cell sorted 5F3⁺ cells.

FIG. 3 is a dot plot of fluorescence activated cell sorting (FACS) datashowing the isolation of human CNS neural stem cells using cell surfacemarkers using the monoclonal antibody 5E12. The x axis represents cellstaining for antibodies to CD34 and CD45. The y axis represents cellstaining with the 5E 12 antibody.

FIG. 4 is a two panel dot plot of FACS sorting data showing theisolation of human neural stem cells by cell surface markers. Humanfetal brain cells were enzymatically dissociated as described. Panel Ashows that 5F3⁺ cells co-express the antigen for the 5E12 antibody.Panel B shows that 5F3⁺ cells typically do not express the antigen forthe 8G1 (CD24) antibody.

FIG. 5 is a chart showing the distribution of 5F3⁺ cells in fetal brainas a function of gestational age.

FIG. 6 is a series of photographs showing results of the transplantationof human neural cells into NOD SCID mouse.

FIG. 7 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the rostral migratory stream (RMS) whentransplanted into a rodent model.

FIG. 8 is a series of photographs showing that the progeny of 5F3⁺sorted cells migrate through the (RMS) into the olfactory bulb whentransplanted into a rodent model.

FIG. 9 shows the characteristics of AC133+ cells. FIG. 9A is a flowcytometric separation of fresh fetal brain cells based on AC133expression. Human fetal brain cells were enzymatically dissociated.Cells were stained with mAbs against CD34, CD45 and AC133, and separatedinto AC133⁻ and AC133⁺ fractions. The residual hematopoietic andendothelial cells were excluded by gating CD45⁻ and C34⁻, respectively.The sorted AC133⁻ (FIG. 9B) and AC133⁺ (FIG. 9C) subsets were culturedin the serum free media and the proliferation of these sorted cells weremonitored for 15 days.

FIG. 10 shows a quantitative analysis of NS-IC activity by limitingdilution and single cell sorting. FIG. 10A is a quantitative analysis ofNS-IC activity by limiting dilution analysis after cell sorting. Sortedcells were plated in a series of limiting cell doses into 96 well-platesby the FACS-ACDU. FIG. 10B shows the clonal expansion of neuralstem/progenitor cells. Neurospheres can be derived from single sortedAC133⁺ cell directly isolated from fetal brain or from a single AC133⁺cell from sorted/expanded cultured neurospheres. FIG. 10C shows thedifferentiation capacity of clonally derived neurosphere cells. Progenyof single cell-derived neurospheres can be differentiated into neurons(-tubulin, green) and astrocytes (GFAP, red).

FIG. 11 shows the detection of human neural cells in non-neurogenicsites. In some cases, human neural cells injected into neonatal brain ofNOD-SCID mice were detected in corpus callosum, cerebral cortex andcerebellum.

FIG. 12 shows the in vivo long-term proliferation of progeny oftransplanted human neurosphere cells in the SVZ of NOD-SCID mice.AC133⁺-sorted/expanded human neurosphere cells were transplanted intothe lateral ventricles of neonatal NOD-SCID mice. Engraftment of humancells were analyzed 7 months after transplantation. FIG. 12A is aschematic diagram of adult mouse brain. Sites where human cellengraftment was evaluated are noted in the diagram. SVZ, subventricularzone; RMS, rostral migratory stream. FIG. 12B shows the detection ofhuman neural cells in the SVZ. A sagittal section of the transplantedmouse brain was stained with anti-human nuclear antigen (FITC, green)and GFAP (Cy-5, blue). FIG. 12C shows the detection of proliferatinghuman neural cells in the SVZ. The sagittal section of the transplantedmouse brain was stained with anti-human nuclear antigen (FITC, green),Ki67 (Cy-3, red) and GFAP (Cy-5, blue). Most of human nuclear antigenpositive cells in the SVZ also co-expressed the proliferation marker,Ki67.

FIG. 13 shows the in vivo migration and differentiation of transplantedhuman neurosphere cells into the RMS and olfactory bulb.AC133⁺-sorted/expanded human neurosphere cells were transplanted intolateral ventricle of neonatal NOD-SCID. Engraftment of human cells wereanalyzed 7 months after transplantation. FIG. 13A shows the progeny ofAC133⁺ sorted/expanded neurosphere cells migrate through the RMS. In theRMS, the array of human nuclear antigen⁺ cells, was also positive withHoechst 33234 counter staining (pink) (i). The human nuclear antigenpositive cells (Cy-3, red) in the RMS were co-localized with β-tubulinexpression (Alexia 488, green) (ii). Some of these cells were clearlydouble positive (ii, arrow). In a different section, cells in the RMSwere stained with human specific N-CAM (FITC, green) and GFAP (Cy-5,blue) (iii) FIG. 13B shows the migration and differentiation of humanneural cells into the olfactory bulb. In the olfactory bulb, humannuclear antigen positive cells were distributed into the glomerularlayer of the olfactory bulb (i and ii). In some cases, human N-CAM⁺neuronal cells were detected (iii).

FIG. 14 shows the in vivo long-term proliferation and differentiation ofprogeny of transplanted human neurosphere cells in the dentate gyrus ofhippocampus. AC133⁺-sorted/expanded human neurosphere cells weretransplanted into the lateral ventricle of neonatal NOD-SCID mice. Sevenmonths after transplantation of AC133⁺-sorted/expanded human neurospherecells into the lateral ventricles of neonatal NOD-SCID miceproliferating human cells can be found in the hippocampus. FIG. 14Ashows the detection of proliferating human neural cells in thesubgranular zone of the dentate gyrus. The sagittal section of thetransplanted mouse brain was stained with anti-human nuclei (FITC,green), Ki67(Cy-3, red) and GFAP (Cy-5, blue). Some human nuclearantigen⁺ cells were co-stained with Ki 67 (arrow). FIG. 14B shows thedetection of human neurons in the dentate gyrus. The sagittal section ofthe transplanted mouse brain was stained with anti-human nuclear antigen(Cy-3, red) and -tubulin (Alexia 488, green). One of two human nuclearantigen-positive cells was also positive for -tubulin (arrow).

DETAILED DESCRIPTION OF THE INVENTION

A population of cells exists within the adult central nervous system(CNS) which exhibit stem cell properties, in their ability to self-renewand to produce the differentiated mature cell phenotypes of the adultCNS. These stem cells are found throughout the CNS, and particularly inthe subventricular regions, and dentate gyms of the hippocampus.

Growth factor-responsive stem cells can be isolated from many regions ofthe neuraxis and at different stages of development, of murine, rodentand human CNS tissue. These cells vary in their response to growthfactors such as EGF, basic FGF (bFGF, FGF-2) and transforming growthfactor alpha (TGF∝), and can be maintained and expanded in culture in anundifferentiated state for long periods of time. Both adult andembryonic murine progenitor cells respond to EGF and grow as spheres ofundifferentiated cells. These cells show the characteristics of stemcells in that they are multipotent, and under serum containingconditions can differentiate into neurons, astrocytes andoligodendrocytes, as well as maintaining a subpopulation which remainsundifferentiated and continues to proliferate under EGF administration.Murine EGF-responsive progenitor cells express mRNA for the EGF receptorin vitro. Human CNS neural stem cell cultures have also been identified.The identification, culture, growth, and use of mammalian, includinghuman, neural stem cell cultures, either as suspension cultures or asadherent cultures, is disclosed in Weiss et al., U.S. Pat. No. 5,750,376and Weiss et al., U.S. Pat. No. 5,851,832, both incorporated herein byreference. Similarly, Johe, U.S. Pat. No. 5,753,506, incorporated hereinby reference, refers to adherent CNS neural stem cell cultures. Whencultured in suspension, CNS neural stem cell cultures typically formneurospheres.

FIG. 1 is shows the proliferation of a NS-IC as it develops into aneurosphere, and subsequent differentiation into neuronal and glialphenotypes, as well as generation of a progeny NS-IC. In the presence ofone or more proliferation-inducing growth factors, the NS-IC divides andgives rise to a sphere of undifferentiated cells composed of more stemcells and progenitor cells (a “neurosphere”). When the clonally derivedneurosphere is dissociated and plated as single cells, in the presenceof one or more proliferation-inducing growth factors, each NS-IC cangenerate a new neurosphere. The cells of a single neurosphere are clonalin nature because they are the progeny of a single neural stem cell. Inthe continued presence of a proliferation-inducing growth factor such asEGF or the like, precursor cells within the neurosphere continue todivide resulting in an increase in the size of the neurosphere and thenumber of undifferentiated neural cells. The neurosphere is notimmunoreactive for glial fibrillary acidic protein (GFAP; a marker forastrocytes), neurofilament (NF; a marker for neurons), neuron-specificenolase (NSE; a marker for neurons) or myelin basic protein (MBP; amarker for oligodendrocytes). However, cells within the neurosphere areimmunoreactive for nestin, an intermediate filament protein found inmany types of undifferentiated CNS cells (Lehndahl et al., 60 Cell585–595 (1990), incorporated herein by reference). Antibodies areavailable to identify nestin, including the rat antibody referred to asRat401. If the neurospheres are cultured in conditions that allowdifferentiation, the progenitor cells differentiate to neurons,astrocytes and oligodendrocytes. The mature phenotypes associated withthe differentiated cell types that may be derived from the neural stemcell progeny are predominantly negative for the nestin phenotype.

The terminology used for undifferentiated, multipotent, self-renewing,neural cells has evolved such that these cells are now termed “neuralstem cells.” A neural stem cell is a clonogenic multipotent stem cellwhich is able to divide and, under appropriate conditions, hasself-renewal capability for NS-IC and can include in its progenydaughter cells which can terminally differentiate into neurons,astrocytes, and oligodendrocytes. Hence, the neural stem cell is“multipotent” because stem cell progeny have multiple differentiationpathways. A neural stem cell is capable of self maintenance, meaningthat with each cell division, one daughter cell will also be on averagea stem cell.

The non-stem cell progeny of a neural stem cell are typically referredto as “progenitor” cells, which are capable of giving rise to variouscell types within one or more lineages. The term “neural progenitorcell” refers to an undifferentiated cell derived from a neural stemcell, and is not itself a stem cell. Some progenitor cells can produceprogeny that are capable of differentiating into more than one celltype. For example, an O-2A cell is a glial progenitor cell that givesrise to oligodendrocytes and type II astrocytes, and thus could betermed a “bipotential” progenitor cell. A distinguishing feature of aprogenitor cell is that, unlike a stem cell, it does not exhibit selfmaintenance, and typically is thought to be committed to a particularpath of differentiation and will, under appropriate conditions,eventually differentiate into glia or neurons.

The term “precursor cells” refers to the progeny of neural stem cells,and thus includes both progenitor cells and daughter neural stem cells.

Cell markers. This invention provides for the identification, isolation,enrichment, and culture of neural stem cells that are capable of formingneurospheres (NS-IC). NS-ICs are identified or selected through thebinding of antigens, found on the surfaces of NS-ICs, to reagents thatspecifically bind the cell surface antigen.

One of these antigens is an antigen that binds to the AC133 monoclonalantibody. The AC133 antibody (herein referred to as the 5F3 antibody) isexemplary of antibody embodiments of reagents that recognize a humancell marker termed prominin. Prominin is a polytopic membrane proteinexpressed in various epithelial cells (Weigmann et al., 94(23) Proc.Natl. Acad. Sci. USA. 12425–30 (1997); Corbeil et al., 112 (Pt 7) J.Cell. Sci. 1023–33 (1999); Corbeil et al., 91(7) Blood 2625–6 (1998);Miriglia et al., 91(11) Blood 4390–1 (1998)). Various AC133 antibodiesare described in U.S. Pat. No. 5,843,333, incorporated herein byreference. A deposit of the murine hybridoma cell line AC133 was made atthe American Type Tissue Collection, 12301 Parklawn Drive, Rockville Md.20852, on Apr. 24, 1997, and given the ATCC designation HB12346. TheseAC133 antibodies are capable of immunoselection for the subset of humancells of interest in this invention. Preferred AC133 monoclonalantibodies can be obtained commercially from Miltenyi Biotec Inc.(Auburn Calif.), including AC133/1-PE antibody (Cat #808-01) andAC133/2-PE antibody (Cat #809-01). For MACS separation, a 50:50 mixtureof the monoclonal antibodies is preferred. The high tissue specificityof AC133 expression is particularly advantageous during enrichment forhighly purified NS-IC populations.

5E12 is a novel monoclonal antibody generated againstenzymatically-dissociated human fetal brain cells. The 5E12 monoclonalantibody was generated substantially according to the contralateralimmunization method described in Yin, U.S. Pat. No. 5,843,633,incorporated herein by reference. The antigen to which 5E12 binds has aputative MW 125 kD, and is currently believed to be a distinct antigenfrom prominin.

CD45 is the T200/leucocyte common antigen. Antibodies to CD45 arecommercially available. In a preferred embodiment, the cells of thisinvention and cultures containing them, are additionally characterized(in addition to being prominin positive) as lacking cell surface markerssuch as CD45.

CD34 is also known as gp105–120. Monoclonal antibodies to CD34 arecommercially available, and CD34 monoclonal antibodies have been used toquantitate and purify lymphohematopoietic stem/progenitor cells forresearch and for clinical bone marrow transplantation.

The monoclonal antibody 8G1 is believed to recognize CD24 (antibodies toCD24 are commercially available), and specifically reacts with the 515kilodalton -chain of human LRP/A2MR which is expressed in a restrictedspectrum of cell types. A strong immunohistochemical reaction is seen inhepatocytes, tissue macrophages, subsets of neurons and astrocytes inthe central nervous system, fibroblasts, smooth muscle cells, andmonocyte-derived foam cells in atherosclerotic lesions in the arterialwall. The antibody can also be used for the characterization of a subsetof myelomonocytic subtypes of chronic and acute leukemia (CD91).Antibodies to CD91 are commercially available.

Cell deposit. The 5E12.5 and 8G1.7 subject cultures are deposited underconditions that ensure that access to the cultures will be availableduring the pendency of the patent application disclosing them to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. The deposits areavailable as required by foreign patent laws in countries wherecounterparts of the subject application, or its progeny, are filed.However, the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Further, the 5E12.5 and 8G1.7 subject culture deposits will be storedand made available to the public in accord with the provisions of theBudapest Treaty for the Deposit of Microorganisms, i.e., they will bestored with all the care necessary to keep them viable anduncontaminated for a period of at least 30 years after the date ofdeposit or for the enforceable life of any patent which may issuedisclosing the cultures plus 5 years after the last request for a samplefrom the deposit. The depositor acknowledges the duty to replace thedeposits should the depository be unable to furnish a sample whenrequested, due to the conditions of the deposits. All restrictions onavailability to the public of the subject culture deposits will beirrevocably removed upon granting of a patent disclosing them.

Isolation, enrichment, and selection of cells. The population of cellsfrom which NS-ICs are isolated can be a neural tissue, a population ofcells is dissociated from neural tissue, or a population of cells incell culture, e.g., a cells in a neurosphere culture or an adherentneural stem cell culture.

The invention provides for the isolation and identification of NS-ICs.Identification of a neurosphere initiating stem cell (NS-IC) involvescontacting a population of neural cells (or which contains neural orneural derived cells) with a reagent that binds to AC133 antigen anddetecting the contact between the reagent that binds to AC133 antigenand the AC133 antigen on the surface of cells. Those cells to which thereagent binds are identified as NS-ICs. The identity of those cells canbe confirmed by assays to demonstrate that the cells are in fact NS-ICs,capable of neurosphere initiation, self renewal and multipotentcy.

The methods of this invention can also be used to isolate AC133⁺ cellsfrom AC133⁻ cells using an AC133 antibody, by combining a population ofneural cells which contains a fraction of NS-ICs with a reagent thatspecifically binds to the AC133 antigen, and then selecting for AC133⁺cells, to produce a selected population enriched in AC133⁺ NS-ICs ascompared with the population of neural cells before selection.

Accordingly, the invention further provides for the enrichment of NS-ICsfrom neural tissue or neural stem cell cultures (e.g., neurospheresuspension cultures or neural stem cell adherent cultures). Theinvention is thus useful for the enrichment of NS-IC from neural tissuein which stem cells and progenitor cells occur at low frequency, or mayhave been depleted, such as late embryo, juvenile, and adult tissue. Oneof skill in the art can combine a population of neural cells containinga fraction of NS-ICs with a reagent that specifically binds to the AC133 antigen; and select for the AC133⁺ cells. In this way, the selectedAC133⁻ cells are enriched in the fraction of NS-IC as compared with thepopulation of neural cells.

The cell selection can be by any suitable means known in the art,including flow cytometry, such as by fluorescence activated cell sortingusing a fluorochrome conjugated AC133 antibody. The selection can alsobe by high gradient magnetic selection using AC133 antibody isconjugated to magnetic particles. Any other suitable method includingattachment to and disattachment from solid phase, is also contemplatedwithin the scope of the invention.

One of skill in the art can derive the population of cells byimmunoselection using an AC133 antibody. The population of cells shouldcontain at least 30% AC133⁺ NS-ICs, preferably at least 50–70% AC133⁺NS-ICs, and more preferably greater than 90% AC133⁺ NS-ICs. Mostpreferable would be a substantially pure population of AC133⁺ NS-ICs,comprising at least 95% AC133⁺ NS-ICs. The degree of enrichmentobtained, and actually used, depends on a number of factors, includingthe method of selection, the method of growth, and the cell dose of thecells that are placed in culture for the initiation of neurospheres.

The population of cells can be derived from late embryo, juvenile, oradult mammalian central nervous system (CNS) tissue, or it may bederived from existing cultures of neural stem cells, as described inWeiss, U.S. Pat. No. 5,750,376, or Johe, U.S. Pat. No. 5,753,506. In themost preferred embodiment, the NS-IC are human. In some embodiments, theAC133⁺ cells in the population can be complexed to endothelial cells.

The in vitro cell cultures described herein containing an enrichedpopulation of AC133⁺ NSICs are generally characterized in that thecultures stain positive for nestin and, in the presence ofdifferentiation-inducing conditions, produce progeny cells thatdifferentiate into neurons, astrocytes, and oligodendrocytes.

One of skill in the art can introduce an isolated AC133⁺ cell to aculture medium, proliferate the isolated AC133⁺ cell in culture;particularly as a neurosphere; culture the progeny of the isolatedAC133⁺ cell under conditions in which the isolated AC133⁺ celldifferentiates to neurons, astrocytes, and oligodendrocytes; then detectthe presence of neurons, astrocytes, and oligodendrocytes. The presenceof neurons, astrocytes, and oligodendrocytes characterizes the isolatedAC133⁺ cell as an NS-IC.

Typically AC133⁺ NS-IC is cultured in a medium that permits the growthand proliferation of neurospheres. The culture in which the isolatedAC133⁺ cell proliferates can be a serum-free medium containing one ormore predetermined growth factors effective for inducing multipotentneural stem cell proliferation. The culture medium can be supplementedwith a growth factor selected from leukocyte inhibitory factor (LIF),epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2;bFGF) or combinations thereof. The culture medium can be furthersupplemented with neural survival factor (NSF) (Clonetics, CA). Theconditions in which the AC133⁺ cell differentiates to neurons,astrocytes, and oligodendrocytes can be culturing the AC133⁺ cellprogeny on a laminin-coated surface in culture medium containing fetalbovine serum (FBS) without EGF, FGF-2 or LIF.

The invention also provides a method for identifying the presence of agrowth factor that affects the growth of NS-IC. One of skill in the artcombines a composition suspected of containing at least one growthfactor that affects the growth of NS-IC with a composition comprisingNS-IC, then determines the growth of the NS-IC as a function of thepresence of the composition. Altered (increased, decreased, etc.) NS-ICgrowth indicates the presence in the composition of a growth factor thataffects the growth of NS-IC. One can then further identify the growthfactor.

Antibodies to AC133. Antibodies to AC133 may be obtained or prepared asdiscussed in U.S. Pat. No. 5,843,633, incorporated herein by reference.The AC133 antigen can be contacted with an antibody, such as variousAC133 monoclonal antibodies, which have specificity for the AC133antigen. An AC133 antibody is characterized by binding to the AC133protein under Western blot conditions from reducing SDS-PAGE gels. TheAC133 antigen has a molecular weight, based on commercially availablestandards, in the range of about 117 kDa. The AC133 antigen is expressedon a subset of progenitor cells derived from human bone marrow, fetalbone marrow and liver, cord blood, and adult peripheral blood.

Antibodies to AC133 antigen can be obtained by immunizing a xenogeneicimmunocompetent mammalian host (including murine, rodentia, lagomorpha,ovine, porcine, bovine, etc.) with human progenitor cells. The choice ofa particular host is primarily one of convenience. A suitable progenitorcell population for immunization can be obtained by isolating CD34⁺cells from cytokine mobilized peripheral blood, bone marrow, fetalliver, etc. A suitable progenitor cell population for immunization canbe obtained from CNS neural stem cells or other NS-IC. Immunizations areperformed in accordance with conventional techniques, where the cellsmay be injected subcutaneously, intramuscularly, intraperitoneally,intravascularly, etc. Normally, from about 10⁶ to 10⁸ cells will beused, which may be divided up into 1 or more injections, usually notmore than about 8 injections, over a period of from about one to threeweeks. The injections may be with or without adjuvant, e.g. complete orincomplete Freund's adjuvant, specol, alum, etc.

After completion of the immunization schedule, the antiserum may beharvested in accordance with conventional ways to provide polygonalantisera specific for the surface membrane proteins of progenitor cells,including the AC133 antigen. Lymphocytes are harvested from theappropriate lymphoid tisue, e.g. spleen, draining lymph node, etc., andfused with an appropriate fusion partner, usually a myeloma line,producing a hybridoma secreting a specific monoclonal antibody.Screening clones of hybridomas for the antigenic specificity of interestis performed in accordance with conventional methods.

AC 133 antibodies can be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al., 269 J. Biol. Chem. 26267–73 (1994), incorporated herein byreference, and others. DNA sequences encoding the variable region of theheavy chain and the variable region of the light chain are ligated to aspacer encoding at least about 4 amino acids of small neutral aminoacids, including glycine or serine. The protein encoded by this fusionallows assembly of a functional variable region that retains thespecificity and affinity of the original antibody.

AC133 antibodies can be produced by use of Ig cDNA for construction ofchimeric immunoglobulin genes (Liu et al., 84 Proc. Natl. Acad. Sci.3439 (1987) and 139 J. Immunol. 3521 (1987), incorporated herein byreference. mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al., Sequences of Proteins of Immunological Interest. N.I.H.publication No. 91-3242 (1991). Human C region genes are readilyavailable from known clones. The chimeric, humanized antibody is thenexpressed by conventional methods.

AC133 antibodies can be produced as antibody fragments, such as Fv,F(ab′)₂ and Fab. Antibody fragments may be prepared by cleavage of theintact protein, e.g. by protease or chemical cleavage. Alternatively, atruncated gene is designed. For example, a chimeric gene encoding aportion of the F(ab′)₂ fragment would include DNA sequences encoding theCH 1 domain and hinge region of the H chain, followed by a translationalstop codon to yield the truncated molecule.

Immunostaining Biological samples are assayed for the presence of AC133⁺cells by any convenient immunoassay method for the presence of cellsexpressing the surface molecule bound by the subject antibodies. Assaysmay be performed on cell lysates, intact cells, frozen sections, etc.The antibodies available from Miltenyi Biotec Inc. (Auburn Calif.) aresuitable for the direct immunofluorescent staining of cells.

Cell sorting. The use of cell surface antigens to NS-IC cells provides ameans for the positive immunoselection of progenitor cell populations,as well as for the phenotypic analysis of progenitor cell populationsusing flow cytometry. Cells selected for expression of AC133 antigen maybe further purified by selection for other stem cell and progenitor cellmarkers.

For the preparation of substantially pure progenitor and stem cells, asubset of progenitor cells is separated from other cells on the basis ofAC133 binding. Progenitor and stem cells may be further separated bybinding to other surface markers known in the art.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography and “panning”with antibody attached to a solid matrix, e.g. plate, or otherconvenient technique. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Dead cellsmay be eliminated by selection with dyes associated with dead cells(propidium iodide [PI], LDS). Any technique may be employed which is notunduly detrimental to the viability of the selected cells.

Conveniently, the antibodies are conjugated with labels to allow forease of separation of the particular cell type, e.g. magnetic beads;biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry. Multi-color analysis is of interest for the separation ofcells based on multiple surface antigens, e.g. AC133⁺ CD45⁻, AC133⁻CD34⁺, etc. Fluorochromes which find use in a multi-color analysisinclude phycobiliproteins, e.g. phycoerythrin and allophycocyanins;fluorescein and Texas red. A negative designation indicates that thelevel of staining is at or below the brightness of an isotype matchednegative control. A dim designation indicates that the level of stainingmay be near the level of a negative stain, but may also be brighter thanan isotype matched control.

In one embodiment, the AC133 antibody is directly or indirectlyconjugated to a magnetic reagent, such as a superparamagneticmicroparticle (microparticle). Direct conjugation to a magnetic particleis achieved by use of various chemical linking groups, as known in theart. Antibody can be coupled to the microparticles through side chainamino or sufhydryl groups and heterofunctional cross-linking reagents. Alarge number of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyidithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

Alternatively, AC133 antibody is indirectly coupled to the magneticparticles. The antibody is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available.

To practice the method, the ACI 33 antibody is added to a cell sample.The amount of AC133 Ab necessary to bind a particular cell subset isempirically determined by performing a test separation and analysis. Thecells and AC133 antibody are incubated for a period of time sufficientfor complexes to form, usually at least about 5 min, more usually atleast about 10 min, and usually not more than one hr, more usually notmore than about 30 min.

The cells may additionally be incubated with antibodies or bindingmolecules specific for cell surface markers known to be present orabsent on progenitor or stem cells.

The labeled cells are separated in accordance with the specific antibodypreparation. Fluorochrome labeled antibodies are useful for FACSseparation, magnetic particles for immunomagnetic selection,particularly high gradient magnetic selection (HGMS), etc. Exemplarymagnetic separation devices are described in WO 90/07380,PCT/US96/00953, and EP 438,520. The AC133 Cell Isolation Kit (MiltenyiBiotec Inc., Auburn Calif.) can be used for the positive selection ofAC133⁺ cells. The kit provides a tool for single step isolation ofAC133⁺ cells. The AC133 Cell Isolation Kit contains FcR Blocking Reagentand MACS colloidal MicroBeads conjugated to the monoclonal mouseanti-human AC133 antibody.

The purified cell population may be collected in any appropriate medium.Various media are commercially available and may be used, includingDulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution(HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove'smodified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with5 mM EDTA, etc., frequently supplemented with fetal calf serum (FCS),bovine serum albumin (BSA), human serum albumin (HSA), etc.

Populations highly enriched for human progenitor or stem cells areachieved in this manner. The desired cells will be 30% or more of thecell composition, preferably 50% or more of the cell population, morepreferably 90% or more of the cell population, and most preferably 95%or more (substantially pure) of the cell population.

Use of purified stem cell/progenitor cells. The AC133⁺ stemcells/progenitor cells are useful in a variety of ways. The AC133⁺ cellscan be used to reconstitute a host whose cells have been lost throughdisease or injury. Genetic diseases associated with cells may be treatedby genetic modification of autologous or allogeneic stem cells tocorrect a genetic defect or treat to protect against disease.Alternatively, normal allogeneic progenitor cells may be transplanted.Diseases other than those associated with cells may also be treated,where the disease is related to the lack of a particular secretedproduct such as hormone, enzyme, growth factor, or the like. CNSdisorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include Alzheimer's Disease, MultipleSclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, andParkinson's Disease, have been linked to the degeneration of neuralcells in particular locations of the CNS, leading to the inability ofthese cells or the brain region to carry out their intended function. Byproviding for maturation, proliferation and differentiation into one ormore selected lineages through specific different growth factors theprogenitor cells may be used as a source of committed cells.Neurospheres can also be used to produce a variety of blood cell types,including myeloid and lymphoid cells, as well as early hematopoieticcells (see, Bjornson et al., 283 Science 534 (1999), incorporated hereinby reference).

The AC133⁺ cells may also be used in the isolation and evaluation offactors associated with the differentiation and maturation of cells.Thus, the cells may be used in assays to determine the activity ofmedia, such as conditioned media, evaluate fluids for growth factoractivity, involvement with dedication of lineages, or the like.

The AC133⁺ cells may be frozen at liquid nitrogen temperatures andstored for long periods of time, being thawed and capable of beingreused. The cells will usually be stored in 5% DMSO and 95% fetal calfserum. Once thawed, the cells may be expanded by use of growth factorsor stromal cells associated with stem cell proliferation anddifferentiation.

The following EXAMPLES are presented in order to more fully illustratethe preferred embodiments of the invention. These EXAMPLES should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLE 1 AC133 Magnetic Cell Sorting (MACS)Positive Selected FetalBrain Cells Contain Neurosphere Initiating Cell (NS-IC) Activity

AC133⁺ cells are prepared by the following method: Human fetal brain(FBR 10–20 gestational week [“g.w.”]) were obtained from AdvancedBioscience Resources, INC (Oakland, Calif.) after obtaining informedconsent. Human fetal brain tissues were cut into 1–3 mm cubed piecesusing scalpels, transferred into 50 mL centrifuge tubes and wash oncewith 0.02% EDTA/PBS solution. Tissues were dissociated enzymatically inthe presence of collagenase and hyaluronidase at 37° C. for 1 hr. Debrisand aggregates were removed by filtering cell suspensions through 70micron filter cup.

AC133⁺ human fetal brain cells were separated by using paramagneticantibody microbeads, AC133/1 Cell isolation Kit (Cat. # 508-01, MiltenyiBiotec, Auburn, Calif.). MACS separations were performed based oninstruction accompanied with the kit. In a representative flowcytometric MACS separation from a typical AC133⁺ isolation, about 44% ofthe cells were AC133⁺ CD45⁻, while about 2% were CD34⁺ (These CD34⁻cells were endothelial cells complexed to the purified NS-IC).

The AC133⁺ selected cells which resulted from the method described above(using brain 18 g.w.) were still heterogeneous. The cells tended to forma complex with endothelial cells.

Endothelial cells were identified as CD34⁻ or CD105⁻. AC133 MACSseparation also enriches CD34⁻ endothelial cells which are associatedwith AC133⁺ cells. (After passaging, the NS-IC separate from thecomplexed endothelial cells and purified NS-IC can be obtained.)

AC133⁺ MACS separated cells were cultured in the presence of mediacontaining EGF, FGF-2, and LIF, as described above. In general, cellsfrom early gestational age fetal brain (5–12 g.w.) were enriched forNS-IC and no enrichment was required to initiate neurosphere cultures.On the other hand, cells from older fetal brain samples (16–20 g.w.)contained far less NS-IC activity and required enrichment for initiatingneurosphere cultures. In other words, AC133⁺ is useful for theenrichment of NS-IC from older gestational age humans brain tissue.AC133⁺ MACS separated cells from fetal brain (18 g.w.) were enriched forNS-IC activity, while whole human fetal brain cells (18 g.w.) withoutAC133⁺ MACS separation failed to initiate neurosphere cultures.

Neurosphere cells established from AC 133 MACS cells express nestin, astested after ˜7 days in culture and detected by rabbit anti-human nestinpolyconal antibodies. For example, among the neurosphere cells availablefrom CytoTherapeutics (Sunnyvale, Calif.), FBR 1069 (18 g.w.) and FBR1070 (20 g.w.) expressed nestin. When induced to differentiate, theAC133+MACS-derived neurosphere cells could differentiate into neuronsand astrocytes, as detected by P-tubulin staining for neurons and GFAPstaining for astrocytes. In this particular differentiation assay,neurosphere cells were cultured onto a laminin-coated surface in thepresence of 1% FBS and without EGF, FGF-2 and LIF.

Other differentiation assay can be used to induce differentiation ofNS-IC to neurons, astrocytes and oligodendrocytes.

EXAMPLE 2 AC133 is a Critical Cell Surface Marker Expressed on Cellsfrom Long-Term Neurosphere Culture

A long-term neurosphere cell culture, 8.5 FBR, was obtained fromCytoTherapeutics Inc. (Providence, R.I.). The 8.5 FBR neurosphere cellsexpress AC133 relatively uniformly. These 8.5 FBR cells are also Thy-1⁺,CD166⁺, and HLA-DR⁺. When Ex Vivo 15 was used as basal media, higherpercentage of neurosphere cultures initiated from primary brain tissuefrom 18 g.w. It is therefore possible to evaluate AC133⁺ fraction ofcells in developing neurosphere cultures. The proportion of AC133⁺ cellsincreased as neurosphere developed. Once neurosphere cells were wellestablished, virtually all cells forming neurospheres expressed AC133.

EXAMPLE 3 Neurosphere Initiating Cells can be Separated Using MonoclonalAntibody, AC133; Flow Cytometry Cell Sorting (FACS) Approach

The purpose of this EXAMPLE is to test whether AC133⁺ cells were theonly cells in the brain that have pluripotent NSC activity. mAb againsthuman CD45 was used to exclude blood cell contamination in fetal tissue.In some cases, mAb against human CD34 was also used to excludeendothelial cells and endothelial-neural progenitor complexes. The fetalbrain cells were thus defined as CD45⁻ CD34⁻. To measure neural stemcells and primitive progenitor activities, a NS-IC assay was establishedto determine frequency of NS-IC in a given population. When NS-IC arerare and express AC133 antigen uniformly, NS-IC can be enriched byAC133+selection, and correspondingly depleted in other fractions.

Source of monoclonal antibodies. AC133 antigen was defined by two mAbsAC133/1 and AC133/2, both conjugated with phycoerythrin, which areavailable through Miltenyi Biotec (Auburn, Calif.). Anti human CD45⁻FITCand Glycophrin A-FITC were obtained from CALTAG (Burlingame, Calif.) andCoulter (Miami, Fla.) respectively. Anti-humanallophycocyanin-conjugated CD34 was obtained from BDIS (San Jose,Calif.).

Cell preparation. FBR were dissociated by collagnease and hyaluronidase,and still contained endothelial-progenitor complex, which prevented theisolation of a candidate of NSC in single cell suspension (endothelialcells are CD45⁺). To dissociate this endothelial cell-NS-IC complex, FBRcells processed as described above, were further treated with trypsinfor 10–15 min. The AC133 antigen, CD45 antigen and CD34 antigen wereresistant for trypsin treatment, while Glycophrin A was sensitive.

After trypsin digestion, cells were washed and stained with mAbs againstCD45, Glycophrin A, AC133 and CD34. No immunomagnetic bead selectionswere used. The cells were incubated for 20–60 min on ice. After thefinal wash, the cells were resuspended in HBSS solution containing 1μg/mL propidium idodine (PI). The labeled cells were analyzed and sortedwith a dual-laser FACS (Becton Dickinson, San Jose). Dead cells wereexcluded from analysis by their PI staining characteristics. Aftersorting purity of sorted cell populations were checked by FACSreanalysis. A representative FACS profiles of before sorting andpost-sorting of AC133⁺ CD45⁻ cells (NS-IC, 5% of the starting cells) andAC133⁻ CD45⁻ cells (˜87% of the starting cells) was performed.

NS-IC activity is highly enriched in the AC133⁺ but not the AC133⁻subset. FBR cells (typically 16–20 g.w.) were typically sorted for CD45⁻CD34⁻ AC133⁻ and CD45⁻ CD34⁻ AC133⁺ fractions. No significant NS-ICactivity resided in CD45⁺ or CD45⁻CD34⁺ populations in FBR.

The sorted cells were cultured in the neurosphere media described above.Typically, Ex Vivo 15 or combination of Ex Vivo 15, D-MEM, F-12 mediawere used as a basal medial. To maximize neurosphere development, thesorted cells were typically cultured in the presence of LIF, FGF-2, EGFand neural survival factor, NSF (Cat. CC-4323, Clonetics, San Diego,Calif.).

A single cell suspension was obtained after cell sorting. After 4–5 daysin vitro culture, the AC133⁺ cells started to proliferate and smallneurospheres were observed 8–10 days post culture initiations. The cellscould initiate neurospheres when cultured in the presence of LIF, FGF-2,EGF without NSF. Neurosphere cultures were initiated from four out offour FBR tissues (18–20 g.w.) sorted for AC133⁺ CD45⁻ or AC133⁺ CD45⁻CD34⁻.

In contrast, when AC133⁻ CD45⁻ FBR cells were placed in culture in thepresence of LIF, FGF-2, and EGF, very few neurosphere formations wereseen, and failed to passage to a new flask. When additional NSF wasadded in the growth media, some neurosphere initiation was observed.Thus, AC133⁻ CD45⁻ FBR cells were depleted in a significant amount ofNS-IC.

EXAMPLE 4 AC133⁺ Cell Separation to Enrich for NS-IC Cells

AC133⁺ cell separation can effectively be used to enrich for NS-IC cellsfrom tissue. Furthermore AC 133⁺ cell separation can further enrich forNS-IC cells from established preparations. In one test, AC133⁺ cellsorting of dissociated neurospheres (CytoTherapeutics, Providence, R.I.)provides a greatly enriched NS-IC culture and shows increasedneurosphere establishment. Using that culture, the cell dose required toinitiate a neurosphere in each well (i.e., 100% positive) can be reducedfrom 3,000–10,000 cells to about 30 cells (see, TABLE 1, below).

TABLE 1 Tissue # % % ID Cell Cell Dose Score Well Positive Negative FBR1104 Post trypsin 1,000 6 24 25.0% 75.0% Ex Vivo 15 3,000 20 24 83.3%16.7% LIF/EGF/ 10,000 24 24 100.0% 0.0% FGF-2 30,000 12 12 100.0% 0.0%100,000 12 12 100.0% 0.0% FBR 1104 Post trypsin 1,000 23 24 95.8% 4.2%Ex Vivo 15 3,000 24 24 100.0% 0.0% LIF/EGF/ 10,000 24 24 100.0% 0.0%FGF-2/NSF 30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1104AC133 neg. 1,000 0 24 0.0% 100.0% selected cells Ex Vivo 15 3,000 1 244.2% 95.8% LIF/EGF/ 10,000 28 48 58.3% 41.7% FGF-2/NSF AC133⁺ 10 2 248.3% 91.7% selected cells Ex Vivo 15 100 24 24 100.0% 0.0% LIF/EGF/ 30024 24 100.0% 0.0% FGF-2/NSF 1,000 24 24 100.0% 0.0% FBR 1101 Posttrypsin 1,000 9 24 37.5% 62.5% Ex Vivo 15 3,000 16 24 66.7% 33.3%LIF/EGF/ 10,000 23 24 95.8% 4.2% FGF-2 30,000 12 12 100.0% 0.0% 100,00012 12 100.0% 0.0% FBR 1101 Post trypsin 1,000 16 24 66.7% 33.3% Ex Vivo15 3,000 21 24 87.5% 12.5% LIF/EGF/ 10,000 24 24 100.0% 0.0% FGF-2/NSF30,000 12 12 100.0% 0.0% 100,000 12 12 100.0% 0.0% FBR 1101 AC133⁺ 1 996 9.4% 90.6% selected cells Ex Vivo 15 10 42 60 70.0% 30.0% LIF/EGF/ 3023 24 95.8% 4.2% FGF-2/NSF 100 11 12 91.7% 8.3%

As shown in TABLE 1, the non-enriched fresh brain tissue (“FBR”) usedhere (g.w. 20) may contain NS-IC in such numbers that it requires a celldose of between 3,000 and 10,000 cells to initiate neurospheres in everywell. By using the method of the invention, enriched populations can beobtained, such that a cell dose of 1,000 cells or less is required, andmore preferably an enriched population such that a cell dose of lessthan 100 cells is required. As shown in TABLE 1, enrichment here hasbeen achieved so that a cell dose of only about 30 cells is required perwell to establish a neurosphere culture in each well. TABLE 1 also showsthat when populations are depleted in AC133⁺ cells (FBR 1104 AC133 neg.selected cells), establishment of neurosphere cultures from thosepopulations is markedly reduced.

Quantitative NS-IC assay. To assay for the presence of NS-IC, cellpopulations suspected of containing the multipotent NS-IC are subjectedto clonal development. Cells are grown in proliferation medium to formneurospheres, then induced to differentiate to form neurons, astrocytes,and oligodendrocytes. The presence of neurons, astrocytes, andoligodendrocytes can be shown by immunostaining. For example, neuronsstain for the presence of β-tubulin; astrocytes stain for the presenceof GFAP; and oligodendrocytes stain for the presence of 04.

The quantitative NS-IC assay can be performed on unpurified tissuecells, on AC133+sorted cells, and on clonal neurosphere cell lines.

EXAMPLE 5 Cell Culture Media for Growth and Passage of NS-IC

Weiss et al., U.S. Pat. No. 5,750,376 and Weiss et al., U.S. Pat. No.5,851,832 disclose “culture medium containing one or more predeterminedgrowth factors effective for inducing multipotent neural stem cellproliferation” and “differentiation-inducing conditions” However,different basal media can be used, including, but not limited to:

-   -   D-MEM/F12 (Gibco BRL, Gaithersburg, Md.);    -   Ex Vivo 15 (Bio Whittaker, Walkersville, Md.);    -   Neural progenitor basal media, (Clonetics. San Diego, Calif.);        or    -   combination of the basal media listed above.

A typical media formulation to culture human neurosphere cells isprovided in TABLE 2.

TABLE 2 SERUM-FREE N2/EGF SUPPLEMENTED CULTURE MEDIUM FOR NEUROSPHERESQuantity Reagents 87 ml DMEM/F12 (Gibco lot. 1012915; Cat. No.11330-032) 1 ml N-2 Supplement (Gibco lot 1017018; Cat. No. 17502-014) 1ml 0.2 mg/ml heparin (Sigma lot 28H0320; Cat. No. H-3149) 1 ml 0.2 MGlutamine (JCR lot 7N2320; Cat. No. 59202-77p) 10 ml 3% Glucose (Sigma,lot 37H0841; Cat. No. G-7021) 20 μl 100 μg/ml EGF (R&D lot CE107091;Cat. No. 236-EG) 100 μl 20 μg/ml FGF-2 (Gibco lot KCQ411; Cat. No.13256-029) 100 μl 10 μg/ml LIF (R&D lot OX038021; Cat. No. 250-L)

EGF is added to 100 ml base medium for human neurospheres afterfiltering the medium. EGF is relatively stable in the medium. FGF-2 andLIF are added when medium is ready to use. The final concentrations ofthe supplement reagents are:

5 g/ml Insulin 100 g/ml Human transferrin 6.3 ng/ml Progesterone 16.1g/ml Putrascine 5.2 ng/ml Selenite 20 ng/ml EGF 20 ng/ml FGF-2 10 ng/mlLIF 2 g/ml heparin 2 mM L-glumtamine 6 mg/ml Glucose

The optimization of media formulation permits a higher percentage ofneurospheres initiated from primary brain tissue to be established. Weprefer Ex Vivo 15 media. The optimization of media formulation alsopermits a more consistent growth of neuro spheres. To maximizeneurosphere development, the NS-IC are typically cultured in thepresence of LIF, bFGF, EGF and neural survival factor, NSF (Cat.CC-432′, Clonetics, San Diego, Calif.). In one test, both trypsinizedFBR 1101 neural cells and trypsinized FBR 1104 neural cells(CytoTherapeutics, Sunnyvale, Calif.), show increased growth whencultured in Ex Vivo 15 medium with LIF, bFGF, EGF, and NSF.

EXAMPLE 6 Direct Isolation of Human Neural Stem Cells from Fetal Brainby Cell Sorting

A large source of highly defined engraftable human cells capable ofextensive neuronal regeneration could be an effective therapeuticproduct for the treatment of neurodegenerative disorders. To definereproducible methods for the enrichment of human neural stem cells(NSCs), we have developed and used monoclonal antibodies (mAbs) directedtoward surface markers on human neural cells to identify and purify NSCsby fluorescence activated cell sorting (FACS). Based on FACS andimmunohistochemical analyses, two mAbs, 5F3 and 5E12 were identified.They defined small subsets of fetal brain cells and displayed specificreactivity to cells in the floor plate and ependymal layer of the spinalcord (12 g.w), sites known to contain CNS stem cells. These mAbs, stainless than 5% of FBR cells, and greater than 95% of cells from long-termneurosphere cultures were positive.

As an example, two cell populations 5F3⁺CD34⁻CD45⁻ (5F3⁻) and5F3⁻CD34⁻CD45⁻ (5F3⁻) were sorted and tested for their ability toinitiate neurosphere cultures. The 5F3+subset was highly enriched forneurosphere-initiating cell activity; they proliferated to form smallneurosphere by 8–10 days in culture. In contrast, the sorted 5F3⁻ cellsremained as a single cell suspension, failed to initiate neurospheres,and eventually died. The expanded 5F3⁺ sorted neurosphere cells werepositive for nestin expression, and differentiated into neurons and gliafollowing exposure to differentiation conditions. Using the NOD SCIDmouse, in vivo studies show that at 8 weeks post transplantation the5F3+neurosphere cells can engraft and migrate. These studies show thatwe have identified and enriched human NSCs based on cell surface markersand flow cytometry and demonstrated their activity using in vitro and invivo assays.

In further tests, we examined brain and spinal cord tissues over variousgestational ages. The earlier (5–12 wk gestation) gestational ages havea higher frequency of neurosphere initiating cell (NS-IC) than latergestational ages (16–20 wk gestation). See, e.g., FIG. 5. Directculturing of cells derived from these tissues leads to neurosphereinitiation.

Our data (shown in the TABLE 3 below) demonstrate that cell populationof neural cells enriched for 5F3⁺ cells are enriched for NS-IC activity,as much as 23 fold.

TABLE 3 Population % in brain NS-IC Range Post processed 100 1/8191/304–1435  brain cells (n = 8) 5F3⁻ sorted (n = 6) 95  1/54341/4224–7772 5F3⁺ sorted (n = 6) 4.6 1/36  1/10–74  

Further, as FIG. 2 shows, neurospheres can be derived from single-cellsorted 5F3⁺ cells. We have also demonstrated that self-renewal ofneurosphere cells derived from 5F3⁺ sorted cells can be achieved byre-initiation of neurospheres from single cells (data not shown).Conversely, our data indicates that cell populations depleted of 5F3⁺cells are also depleted for NS-IC activity.

EXAMPLE 7 Isolation of NS-IC by Different Markers

As a second example, we sorted cell populations using a novel monoclonalantibody, 5E 12, described herein. The 5E12⁺ subset was enriched forneurosphere-initiating cell activity, as shown in TABLE 4 below. Seealso FIG. 3. Our data suggests that the antigen to the 5E12 antibody isco-expressed with the AC133 antigen on 5F3⁻ cells.

We also evaluated the 8G1 monoclonal antibody as a subselector forneural stem cells, as shown in TABLE 4 below. Cells that were 5F3⁺ and8G1^(−/lo) displayed more stem cell-like properties, while cells thatwere 5F3⁻ and 8G1^(med/hi) displayed more progenitor cell-likeproperties.

TABLE 4 ENRICHMENT OF NS-IC BY 5F3, 5E12 AND 8G1 ANTIBODIES Population %in brain NS-IC Range Brain cells 100 1/819  1/304–1435  control (n = 8)5F3− sorted (n = 6) 95 1/5434 1/4224–7772 5F3+ (n = 6) 4.6 1/36 1/10–74   5E12− (n = 2) 97 1/1335 1/1259, 1411 5E12+ (n = 3) 2.5 1/286 1/79–392   5F3+ 8G1^(−/lo) (n = 3) 1.1 1/23  1/15–34   5F3+ 8G1^(mid/hi)(n = 3) 1.7 1/63  1/38–105   *All sorted populations were CD34⁻ CD45⁻

EXAMPLE 9 In Vivo Studies NS-IC

We transplanted 5F3⁺-sorted NS-ICs (obtained as described above) intothe lateral ventricles of neonatal immunodeficient mice, usingconventional techniques. Engraftment and migration of human neurospherecells were detected between 4–8 weeks after injection using a humanspecific Thy-1 antibody (see FIG. 6). As shown in FIG. 7, staining withhuman β-tubulin (a neuronal marker) and human nuclear antigen (forlocalization of human cells) revealed migration of the human neurospherecells through the rostral migratory stream (RMS). Further, as shown inFIG. 8, localization using human nuclear antigen demonstrated that humanneurosphere cells had migrated through the RMS to the olfactory bulb.

EXAMPLE 10 Direct Isolation of and Transplantation of Human CentralNervous System Stem Cells

Introduction and summary. Stem cells, clonogenic cells with self-renewaland multilineage differentiation properties, have the potential toreplace or repair damaged tissue. In this EXAMPLE, we have isolatedclonogenic human brain stem cells (CNS-SC) that initiate neurospherecultures and show both self-renewal and differentiation to neurons andglia. These non-genetically modified human CNS-SC are marked bymonoclonal antibodies for surface markers. The cells are 5F3 (AC133)⁺,5E12⁺, CD34⁻CD45^(− l and CD)24^(−/lo). Single AC133⁺CD34⁻CD45⁻ sortedcells initiated neurosphere cultures and displayed multilineagedifferentiation. Once transplanted into brains of immunodeficientneonatal mice the sorted and expanded CNS-SC show potent engraftment,proliferation, migration and differentiation in a site-specific manner.

In this EXAMPLE, we show that mAbs 5F3 and the novel mAb, 5E12, detect adistinct subset of human fetal brain (FBr) cells. Fluorescence-activatedcell sorting using these mAbs in conjunction with mAbs that markcontaminating blood and endothelial cells (CD34 and CD45) results in asubset of human FBr cells, AC133⁺ CD34⁻CD45⁻. After cell sorting, thesorted cells are capable at the single cell level of neurosphereinitiation, self-renewal, and multilineage differentiation, cells thatwe propose as human CNS-SC. These candidate human CNS-SC self-renewedand significantly expanded in neurosphere cultures, and differentiatedin vitro to neurons and glia. The sorted and expanded candidate CNS-SCcan be transplanted into the lateral ventricles of newborn NOD-SCIDmouse brains, where they undergo apparently appropriate site-specificengraftment, continued self-renewal, migration and differentiation forat least 7 months.

Searching for CNS neural stem cell markers; Strategy. We hypothesizedthat candidate CNS-SC markers should be expressed on only a minor subsetof FBr cells. Since there was evidence that the neurosphere culturescontain an enriched population of stem/progenitor cells, we hypothesizedthat the candidate neural stem cell markers should be more abundantlyexpressed on these cells. Thus, we screened through the mAbs usedpreviously to define human hematopoietic stem cells (HSC).

Sorted populations were tested to determine whether they were enrichedfor neurosphere-initiating cells (NS-IC). Any mAb that cleanly separatedFBr into 2 fractions (one fraction that established a neurosphereculture and one that did not) was considered a candidate mAb to helpidentify NS-IC. In the initial screen, mAbs were sought that positivelystained a small fraction of FBr and a large fraction of long-termcultured neurosphere cells, and other mAbs (for negative selection) thatstained most FBr cells but a small fraction of neurosphere cells. Enzymedissociated FBr and long-term cultured neurosphere cells were stainedwith over 50 known mAbs.

A long-term neurosphere culture, 8.5 FBr has been described previously(Carpenter et al., 158 Exp. Neurol. 265–78 (1999)). These cells werecultured in standard human neurosphere media: D-MEM/F12 basal media withN-2 supplement (Gibco), 3% glucose, 0.2M glutamine and 0.2 mg/ml heparinin the presence of FGF-2 (20 ng/mL), EGF (20 ng/mL), and LIF (10 ng/mL).The cultured cells were harvested and dissociated enzymatically in thepresence of collagenase for 5–10 min for passage or trypsinized toobtain single cell suspension for mAbs staining.

The neurosphere cells did not express the vascular and hematopoieticmarkers CD34 or CD45. In contrast, Thy-1, a critical cell surface markerthat identified both mouse and human HSC, was expressed at high levelson virtually all FBr cells and neurosphere cells, and was therefore notuseful. Interestingly, initial antibody screening revealed that anotherHSC marker, AC133, was expressed on only 1–5% of FBr cells derived from16–20 gestational week (g.w.) tissue and on ˜90% of cultured neurospherecells.

Monoclonal antibody AC133 enriches for human CNS-SC. Human FBr tissuewas obtained from the remains of the 16–20 gestation weeks (g.w.)fetuses from Advanced Bioscience Resources, Inc., according to NIHguidelines. FBr tissues were cut into 1–3 mm pieces using scalpels,transferred into 50 mL centrifuge tubes and washed once with 0.02%EDTA/PBS solution. Tissues were dissociated enzymatically in thepresence of 0.1% collagenase (Roche, Indianapolis, Ind.) and 0.1%hyaluronidase (Sigma, St Louis, Mo.) at 37 degrees in HBSS supplementedwith 0.1% BSA, 10 mM HEPES and DNase for 1 hr. To obtain a single cellsuspension, the dissociated FBr cells were further treated with 0.05%trypsin, 53 mM EDTA (Gibco, Grand Island, N.Y.) for 10–15 min. Debrisand aggregates were removed by filtering the cell suspension through a70-micron filter unit.

Single cell suspensions were obtained after collagenase/hyuronidasetreatment followed by trypsinization. Therefore, screening was limitedto trypsin-resistant cell surface epitopes.

Bulk cultures of sorted CD45⁺ or CD34⁺ cells from FBr failed to initiateneurosphere cultures. Thus, both CD34 and CD45 could be used as negativeselectors for human CNS-SC.

To test whether CNS-SC can be isolated based on AC133 expression, humanFBr cells were stained with antibodies to CD34, CD45 and AC133. AC133antigen was defined by two mAbs AC133/1 and AC133/2, both conjugatedwith phycoerthrin (PE), which are available through Milteny Biotec(Auburn, Calif.). Anti-human CD45-FITC, anti human CD34 conjugated withallophycocyannin (APC) were obtained from CALTAG (Burlingame, Calif.)and BDIS (San Jose, Calif.), respectively. The dissociated FBR cellswere incubated with mAbs against CD45-FITC, AC133/1 AC133/2-PE andCD34-APC for 20–60 min on ice. After the final wash, cells wereresuspended in HBSS solution containing 0.5 g/mL: propidium iodine (PI).The labeled cells were analyzed and sorted with a dual-laser Vantage SE(BDIS, San Jose). Dead cells were excluded from analysis by their PIstaining characteristics. Contaminating blood cells and endothelialcells were excluded by gating out CD45+cells CD34⁺ cells, respectively.After sorting purity of sorted cell populations were checked.

Two cell populations, AC133⁻CD34⁻CD45⁻ (AC133) and AC133⁺ CD34⁻CD45⁻(AC133⁺), were sorted and cultured for NS-IC activity. Although AC133expression appeared initially to be a continuum (FIG. 9A), FACSpre-enrichment of AC133⁺ cells followed by a second round of cellsorting revealed a distinct AC133⁺ population (FIG. 9A). In allsubsequent tests, both AC133⁻ and AC133⁺ subsets were sorted twice,resulting in AC133⁻ and AC133⁺ FBr cell fractions with high purity (FIG.9A).

AC133⁺ single cell suspensions cultured under neurosphere conditionsbecame blast-like, adhered onto the plastic plate and began to initiatecell division. After 4–5 days in culture, a large fraction of the AC133⁺cells began to proliferate and to float as a cluster of a few cells.Small neurospheres were observed as early as 7–10 days after cultureinitiation (FIG. 9C). In contrast, the sorted AC133⁻ cells remained as asingle cell suspension, failed to initiate neurospheres, and eventuallydied (FIG. 9B). Thus, NS-IC activity is found in the AC133⁺ but not inthe AC133-subset of CD45⁻CD34⁻FBr cells.

Only AC133⁺ human FBr cells contain NS-IC activity. Since thequalitative NS-IC assay showed a striking difference in proliferationbetween AC133⁻ and AC133⁺ subsets, we wanted to determine the frequencyof NS-IC in unfractionated (i.e., post cell processed) and variousfractionated FBr cell suspensions. Unfractionated FBr cells were platedby limiting dilution (100–10,000 cells/well) into 96-well plates usingthe FACS automatic cell deposition unit (ACDU). These plates werecultured for 6–8 weeks, and wells that contained neurospheres werescored as positive.

The sorted FBr cells were cultured in neurosphere culture in Ex Vivo 15(Bio Whittaker, Walkersvile, Md.) media with N2 supplement (Gibco) inthe presence of FGF-1 (20 ng/mL), EGF (20 ng/mL), LIF (10 ng/mL), NeuralSurvival Factor-1 (Clonetics, San Diego, Calif.) and 60 g/mL N-acetylcystine (Sigma) (Neurosphere-initiation media). The media formulationwas optimized based on the frequency analysis of limiting dilution ofFBr cells. Cultures were fed weekly and passaged when the neurospheresbecame large. In some cases, sorted FBr cells were resorted by theautomated cell-deposition unit (ACDU) into 96-well plates to evaluatefrequency of precursors to initiate neurosphere cultures. These 96-wellplates were fed weekly and the presence of neurospheres in wells wasscored 6–8 weeks post culture initiation. Linear regression analysis ofthe proportion of negative wells at each cell concentration was used todetermine the frequency of NS-IC.

Multiple neurospheres were detected in the wells plated with high celldoses (i.e., 10,000 cells per well), whereas in the wells plated with alower cell dose (i.e., 300–1000 cells/well), positive wells containedonly single neurospheres. At 100–300 cells per well, only rare wellscontained a single neurosphere. When the log of negative wells isplotted versus the number of cells plated per well, a straight linefunction was found; at 37% negative wells a single hit for NS-IC wasdetermined (FIG. 10A). In a representative tissue shown in FIG. 10A,control processed brain cells contained NS-IC activity at a frequency of1/880 cells. In the AC 133-subset there was a depletion of NS-ICfrequency to 1/4860 cells. In contrast, NS-IC activity was highlyenriched in the AC133⁺ subset with a frequency of 1/32 cells (FIG. 10A).NS-IC frequency from 8 different FBr tissues (16–20 gestational weeks)were evaluated as summarized in FIG. 10B and TABLE 5.

TABLE 5 ENRICHMENT OF NS-IC ACTIVITY ISOLATED BY CELL SURFACE MARKERSPopulation % in brain NS-IC NS-IC Range Brain cell control 100 1/731 1/139–1435 AC133⁻ sorted* 95 1/4344 1/745–4760 AC133⁺ sorted* 4.6 1/31 1/6–1/74 Brain cell control 100 1/1132 1/661–1435 5E12− sorted* 97 1/1335** 1/1259, 1441 (35672) 5E12+ sorted* 2.5 1/286  1/79–392 Braincell control 100 1/1030 1/399–1435 AC133+ 8G1 ^(−/lo)* 1.1 1/23  1/15–34AC133+ 8G1^(mid/hi)* 1.7 1/63  1/38–105 *All sorted populations werealso gated for CD34⁻ CD45⁻, which represent 98–99% of FBr. **dataexcluding one test, as the actual calculated NS-IC frequency indicatedin the parentheses. It was due to the most of wells were negative.

In unfractionated fetal brain cells, about 1 in 730 cells contains NS-ICactivity. The AC133⁻ subset, representing >95% of FBr contained <1 in4300 cells with NS-IC and was therefore ˜6-fold depleted. In contrast,NS-IC activity was on average enriched 23-fold in the AC133⁺ subset with˜1/31 (range 1/6 to 1/72) fresh FBr cells capable of initiating aneurosphere. Since AC133⁺ cells represent ˜4.6% of FBr (i.e., 1 in 22cells), the enrichment of NS-IC activity virtually paralleled thefrequency of AC133⁺ cells in FBr. Thus, AC133⁺ cells contained alldetectable NS-IC activity in 16–20 gestational week FBr cellsuspensions.

Clonal neurosphere expansion, self-renewal and differentiation fromsingle AC133⁻ sorted cells. To confirm the clonality of neurospheres,single FBr-derived AC133⁺ sorted cells were directly plated into wellsof a 96-well plate by the ACDU. After 8 weeks, some wells containedsingle neurospheres, confirming that these are derived from singleAC133⁺ sorted cells (FIG. 10C).

The self-renewal activity of AC133⁺-derived neurospheres was tested forthe ability to re-initiate neurosphere cultures. For example, primaryneurosphere cultures initiated by AC133⁺-sorted FBr cells weredissociated and re-plated as a single cell per well. Nine 96 wellsshowed development of a single neurosphere. In one test, neurosphereswere clonally derived from AC133⁺-sorted neurosphere cells with about10% seeding efficiency. Consistently, neurosphere cells which becameAC133⁻ failed to re-initiate secondary neurosphere cultures. Thus, thesequantitative results demonstrated that NS-IC activity is highly enrichedin the AC133⁺ and depleted in the AC133⁻ subset of FBr cells.

To test the differentiation capacity of clonally derived neurospherecells, single neurospheres were harvested, plated, and induced todifferentiate in the presence of the neurotrophic factors, BDNF andGDNF. Neurosphere cells were harvested and disssociated with collagenaseand placed onto chamber slides coated with poly-ornithine. They werecultured in the neurosphere-initiation media for one day until they wereseeded on the plate. The culture is replaced with differentiation media,consisted from Ex Vivo-15, N2, NAC, BDNF (10 ng/mL), GDNF (10 ng/mL) andlaminin (1 g/mL). After 1–2 weeks, chamber slides were fixed with 4%paraformaldehyde in PBS and stained to detect differentiation intoneurons and astrocytes, with mAbs against -tubulin and glial fibrillaryacidic protein (GFAP).

Under these conditions, the AC133⁺-derived neurospheres differentiatedinto neurons and astrocytes (FIG. 10D). Together, the data suggest thatthe mAb AC133 is a marker for NS-IC and can be used to enrich for humanCNS-SC. These AC133⁺-derived neurosphere cultures have been continuallypassaged (>P15 prior to freeze). In a rough estimation, the absolutenumber of AC133⁺ cells have increased at least 1000 fold by the 5thpassage (TABLE 6), and they are capable of re-initiating neurospheresreproducibly. Therefore, AC133 defines a neural cell population thatcontinuously expands and self-renews in vitro.

TABLE 6 EXPANSION OF HUMAN AC133⁺-SORTED CELLS Cell number expansionbetween Expansion Days in cultures Passage 0 to 5 Exp. 1 87 924 Exp. 276 944 Exp. 3 96 750 Exp. 4 74 463 Exp. 5 110 2469 Average 89 1013

NS-IC are also enriched in 5E12⁺ and 8G1^(−/lo) cells; Generation ofnovel mAbs. Concurrently with screening the usefulness of availablemAbs, we sought to identify novel cell surface markers on human neuralcells. FBr cells were dissociated with a combination of collagenase andhyluronidase and were used to immunize BALB/c mice, with a decoystrategy to raise tissue-specific mAbs. About 1900 wells growinghybridoma cells were screened and hybridomas were selected, expanded,and further tested for specific reactivity to human brain. Monoclonalantibodies to human neural cells were produced using a decoyimmunization strategy previously described by Yin et al., 90 Blood5002–12 (1997). Briefly, BALB/c mice were immunized in one footpad withdecoy human leucocyte enriched peripheral blood, and in thecontralateral footpad with enzyme dissociated human fetal brain cells.The donor lymph node cells draining the human FBr cells immunizationwere harvested, processed for a cell suspension and fused with a mousemyeloma line. This fusion to a mouse myeloma line has resulted in 1900wells growing hybridoma cells. Approximately 180 hybridomas wereselected, expanded, and further tested for specific reactivity to humanbrain cells.

As described above, the CNS-SC were highly enriched in the AC133⁻subset. To further characterize the phenotype of the CNS-SC, we testedwhether AC133⁺ cells are phenotypically heterogeneous. Thus, AC133⁻cells were screened for the co-expression of other markers from thepanel of new mAbs. Two novel mAbs, 5E12 and 8G1, were identified. Mab5E12 co-stains AC133⁺ cells and long-term neurosphere cells. 5E12⁺ FBrcells are enriched and 5E12⁻ cells are depleted in NS-IC activity (TABLE5).

In developing fetal brain, the majority of FBr cells (>90%) expressed ahigh level of the 8G1 marker (FIG. 4). The AC133⁺ cells wereheterogeneous in 8G1 expression; mostly 8G1^(−/lo), some 8G1^(mid), andfew 8G1^(hi) cells (FIG. 12). Interestingly, 8G1 expression on long-termcultured human neurosphere cells was also heterogeneous.Immunoprecipitation and blocking studies determined that 8G1 is ananti-CD24 mAb. Historically, CD24 is known as the heat stable antigen(HSA) and is used widely as a marker in hemato-lymphopoiesis (Altermanet al., 20 Eur J Immunol 1597–602 (1990)). HSA is expressed at lowlevels on mouse HSCs (Shih & Ogawa, 81 Blood 1155–60 (1993); Spangrude &Scollay, 18 Exp. Hematol. 920–6 (1990)), and at high levels on proB andpre-B cells and thymocytes, and is down regulated completely when thesecells become mature lymphocytes (Alterman et al., 20 Eur. J. Immunol.1597–602 (1990)). As shown in TABLE 5, the frequency of NS-IC is higherin the AC133⁺ CD24^(−/lo) fraction compared to the AC133⁺CD24^(mid/hi)subset.

Potent engraftment ability of AC133⁺-derived human neurosphere cells. Totest the in vivo engraftment, migration and differentiation capacity ofhuman sorted/expanded CNS-SC, 10⁵ or 10⁶ neurosphere cells (initiatedfrom AC133⁺-sorted FBr cells) were injected into the lateral ventriclesof neonatal NOD-SCID mouse brains.

Expanded AC133+ sorted neurosphere cells at passage 6–10 were harvestedand gently dissociated with collagenase to obtain small clusters ofcells. Cells were resuspended in the equivalent of 0.25×10⁵ or 2.5×10⁵/L. Neonatal mice (<24 hrs after birth) were cryo-anesthetized using ice;once anesthetized, the pups were placed on stereotaxic device. A smallhole was drilled in the cartilage with a needle and cell suspensionswere introduced by Hamiliton syringe into both lateral ventricularspaces. Neonatal mice were injected with 2 1 of cells ranging 10⁵–10⁶cells/injection. Pups were revived by warming to monitor the outcome ofsurgery and then returned to the mother. The injected mice were kept for7 months prior to testing engraftment of human cells.

Neonatal NOD-SCID mice were chosen as recipients to maximize theparticipation of injected cells into the neurological system, as cellgenesis is still actively in progress in the some parts of neonatalmouse brain. In addition, immunodeficient mice do not reject humantissues, and SCID and NOD-SCID mice have been used as hosts for in vivostudies of human hematopoiesis and tissue engraftment (McCune et al.,241 Science 1632–9 (1988); Kamel-Reid & Dick, 242 Science 1706–9 (1988);Larochelle et al., 2 Nat. Med. 1329–37 (1996)). Seven months aftertransplantation, animals were sacrificed, and sagittal sections werestained with human specific mAbs against N-CAM and Thy-1 as well ashuman nuclear antigen.

After 7 months post transplantation, NOD-SCID mice were deeplyanesthetized with Avertin and perfused with PBS, followed by 4%paraformaldehycle in phosphate buffer. Brains were post fixed overnight,placed into 30% sucrose solution and frozen in the OCT Compound. Brainswere sectioned sagittally at 5 um thickness for futherimmunocytochemsitry. To detect human cells in the transplanted mousebrains, sections were stained with mouse monoclonal antibodies againsthuman Thy-1 (Pharmingen, San Diego, Calif.), N-CAM, -tubulin (Sigma) ornuclear antigen (Chemicon) followed by goat anti-mouse IG conjugatedwith Alexa 488 (Molecular Probe, Eugene, Oreg.)

Staining of these mAbs on mouse brain controls showed nocross-reactivity. Mabs against GFAP and β-tubulin stained both human andmouse cells, and so double labeling with the anti-human nuclei antibodywas used to demonstrate human lineage-specific cell populations.Detailed analysis focused particularly on the two sites of the brainpreviously shown to be sites of active neurogenesis, the subventricularzone (SVZ) of the lateral ventricles and the dentate gyrus of thehippocampus (FIG. 12A).

Human cells were found throughout the mouse brain and were abundant inthe SVZ seven months after injection (FIG. 12). For example, in one areamany cells with human nuclei+were surrounded by GFAP⁺ cells (FIG. 12B).Confocal microscopic examinations indicated that most of the human cellswere GFAP⁻, but occasional human GFAP⁺ cells were also detected (FIG.12B, arrow).

The transplanted mouse brains were sectioned at 40 micrometer thicknesson a microtome and stained with mAbs, and analyzed using a Bio-Radconfocal scanning light microsocope as described previously by Suhonenet al., 383 Nature 624–7 (1996).

Because stem/progenitor cells in the SVZ have been shown to proliferatecontinuously, we tested whether progeny of the transplanted human AC133⁺sorted/expanded neurosphere cells were still proliferating in situ. Onesection of transplanted brain was stained with Ki67, a marker associatedwith cell proliferation. Strikingly, a cluster of human cells in theSVZ, nested in GFAP⁺ cells, co-express Ki-67, indicating that the humancells in SVZ can, like their mouse counterparts, continue to proliferate7 months post transplantation in the SVZ (FIG. 12C).

In the olfactory system of rodents, the progeny of stem/progenitor cellsthat have proliferated in the SVZ enter the rostral migratory stream(RMS) and migrate to the olfactory bulb (Lois & Alvarez-Buylla, 264Science 1145–8 (1994)). The “chain of neuroblasts” of endogenous rodentprogenitors in the RMS express β-tubulin and N-CAM (Fricker et al., 19J. Neurosci. 5990–6005 (1999); Gage & Cristen, Isolation,characterization and utilization of CNS stem cells (Springer,Heidelberg, 1997)). The sagittal sections containing the RMS wereexamined for the presence of human neural cells. In mice transplantedwith AC133⁺-sorted neurosphere cells, large numbers of human cells weredetected, beginning in the SVZ and extending throughout the RMS (FIG.13A). Many of these clustered human cells were colocalized withβ-tubulin, but it was difficult to identify double positive cells insuch clusters. Careful examination of tissues containing fewer humancells revealed multiple cells that were double positive for bothβ-tubulin and human nuclear antigen (FIG. 13B, arrow). In addition, manyof these cells in the RMS expressed the human specific marker, N-CAM(FIG. 13C).

After migrating though the RMS, the progeny of stem/progenitor cells areexpected to enter the olfactory bulb and extend toward the olfactoryglomerulus to the periglomelular layers of the bulb (Lois &Alvarez-Buylla, 264 Science 1145–8 (1994)). As shown in FIG. 13D, thetransplanted progeny of human cells distributed into the glomerular aswell as the periglomelular layers. Some of these cells expressed humanN-CAM, indicating that they were committed to neuronal lineages. Thus,the progeny of the transplanted sorted/expanded human CNS-SC migrateinto the RMS, distribute and differentiate into neuronal lineage in theolfactory bulb. In a few instances tyrosine hydroxylase positive humancells were observed.

Another site where neurogenesis takes place in adult life is the dentategyrus of the hippocampus (Gage et al., 92 Proc. Natl. Acad. Sci. USA11879–83 (1995)). In this EXAMPLE, we found numerous human nuclei cellsin the dentate gyrus of the hippocampus. Some of the human cells in thesubgranular cell zone co-express Ki-67, indicating that they are stillable to proliferate after 7 months post transplantation (FIG. 14A). In adifferent analysis, some other human cells were β-tubulin⁺, as expectedfor developing granular neurons, with their processes extending towardsthe hilus of the hippocampus (FIG. 14B). These results indicate that notonly do these sorted/expanded human CNS-SC engraft, migrate, continue toproliferate, and differentiate, but also their behavior and cell fatewere regulated by host cues in a site-specific manner. In no case didthe injected cells form tumors.

Discussion. The purpose of this EXAMPLE was to find surface markers onhuman brain cells that allow the prospective isolation of candidatehuman CNS-SC. We hypothesized that human neurosphere cultures might beclonally initiated from CNS-SC, and undertook to isolate the NS-IC fromfresh human FBr cell suspensions. We demonstrated that 16–20 g.w. humanFBr cells contain a rare subset of clonogenic AC133⁺, 5E12⁺, 8G1^(−/lo)CD34⁻CD45⁻ cells that can initiate neurosphere cultures, self-renewextensively in these cultures, and differentiate in vitro to neurons andglia. Upon transplantation to the lateral ventricles of immunodeficientnewborn mice these cells engraft for at least 7 months and give rise toprogeny that differentiate, migrate, and some that continued toproliferate (i.e. presence of Ki-67⁺ human neural cells in the SVZ anddentate gyrus), The engrafted human cells are found in sites whereintheir counter-part endogenous mouse neural cells undergo the sameevents.

The data in FIG. 10 and TABLE 5 demonstrate that there are no otherdetectable NS-IC cells outside of AC133⁺ subset in the human fetalbrain.

During the analysis of AC133 staining patterns, we noted that ˜0.2% ofFBr cells express CD34 and remained adherent to cells that were AC133⁺CD34⁻. These CD34⁺ cells co-expressed the known endothelial cell markersCD105 and CD31 and, after trypsinization, remained as complexes of cellsthat could inefficiently form some neurospheres when plated at highdensity in bulk cultures. We have observed similar complexes after apositive selection for CD34-expressing cells. Thus, there is a frequentassociation of AC133⁺ CNS-SC and CD34⁻ endothelial cells that naturallyexists. The finding that AC133⁺ CNS-SC are perivascular, fits with thehypothesis that neurogenesis proceeds pari-passu with angiogenesis.

It is striking that the candidate human fetal CNS-SC engraft followinginjection into the lateral ventricles of neonatal mice and extendedthroughout the mouse brain. This is a stage of continuing neurogenesisin the mouse, although the extent to which these cells distribute isgreater than what one might have expected. In particular, human cellswere found adjacent to ventricular spaces, in the hippocampus, in thecerebral cortex, in the corpus callosum, as well as in the cerebellarareas of the brain (FIG. 11). In additions, cells are found both in theSVZ (the area of self-renewal and replication) and along the RMS (thearea of differentiation and migration) as well as directly within theolfactory bulb in and near the olfactory glomeruli. Therefore, theinjected human CNS-SC are continuing in the processes of neurogenesis atthese sites.

Snyder et al. using v-myc immortalized mouse and human neural celllines, demonstrated this wide (“global”) distribution of cells (Yandavaet al., 96 Proc Natl Acad Sci USA 7029–34 is (1999)). Here we show thatthis is a property not only of myc-immortalized cells of murine originbut also of nongenetically modified human CNS-SC.

In contrast to transplantation with teratocarcinoma cells (Trojanowskiet al., 122 Exp Neurol 283–94 (1993)), CNS-SC cells do not formneoplastic or hyperplastic aggregates, even when tested 7 months afterinjection in a fully permissive microenvironment. Therefore CNS-SC cellscan reveal developmental and functional pathways inherent in humanneuronal systems not modified by the functions of oncogenes or alreadyneoplastically transformed cell lines. The isolation of CNS-SC providesseveral opportunities for scientific discovery: (i) to delineatedirectly the lineages that derive from cells at a particular stage ordifferentiation; (ii) to obtain a gene expression profile of CNS-SC, andtheir immediate downstream progeny in vitro or in vivo;

-   -   (iii) to test whether specific gene modification of the cells        allows their engraftment, migration, differentiation, and/or        functional integration. The isolation of human CNS stem cells        allows for the transplantation of these cells in mouse analogues        of human disease, as preclinical tests for their        transplantability, differentiation ability, and lack of        tumorigenesis.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

1. A method for producing a population highly enriched for human centralnervous system stem cells (CNS-SC) which can initiate neurospheres(NS-IC), comprising: selecting from a population containing neural orneural-derived cells for cells that bind to a first antibody selectedfrom the group consisting of monoclonal antibody AC133 and monoclonalantibody 5E12, and further comprising the step of further enriching thepopulation by selecting and eliminating from the population those cellsthat bind to a second antibody selected from the group consisting of amonoclonal antibody that binds to CD45 antigen and a monoclonal antibodythat binds to CD34 antigen, such that those cells that are AC133⁺CD45⁻or AC133⁺CD34⁻ or 5E12⁺CD45⁻ or 5E12⁺CD34⁻ are selected.
 2. The methodof claim 1, wherein said first antibody is monoclonal antibody AC133. 3.The method of claim 1, wherein said first antibody is monoclonalantibody 5E12.
 4. The method of claim 1, wherein said second antibody isa monoclonal antibody that binds to CD45 antigen.
 5. The method of claim1, wherein said second antibody is monoclonal antibody that binds toCD34 antigen.
 6. A method for producing a population highly enriched forhuman central nervous system stem cells (CNS-SC) which can initiateneurospheres (NS-IC), comprising: selecting from a population containingneural or neural-derived cells for cells that bind to monoclonalantibody AC133 and monoclonal 5E12, and further comprising the step offurther enriching the population by selecting and eliminating from thepopulation those cells that bind to a monoclonal antibody that binds toCD45 antigen or monoclonal antibody that binds to CD34 antigen, suchthat those cells that are AC133⁺5E12⁺CD45⁻ or AC133⁺5E12⁺CD34⁻ areselected.
 7. A method for producing a population highly enriched forhuman central nervous system stem cells (CNS-SC) which can initiateneurospheres (NS-IC), comprising: selecting from a population containingneural or neural-derived cells for cells that bind to monoclonalantibody AC133 or monoclonal 5E12, and further comprising the step offurther enriching the population by selecting and eliminating from thepopulation those cells that bind to a monoclonal antibody that binds toCD45 antigen and a monoclonal antibody that binds to CD34 antigen, suchthat those cells that are AC133⁺CD45⁻CD34⁻ or 5E12⁺CD45⁻CD34⁻ areselected.
 8. A method for producing a population highly enriched forhuman central nervous system stem cells (CNS-SC) which can initiateneurospheres (NS-IC), comprising: selecting from a population containingneural or neural-derived cells for cells that bind to monoclonalantibody AC133 and to monoclonal antibody 5E12, and further comprisingthe step of further enriching the population by selecting andeliminating from the population those cells that bind to a monoclonalantibody that binds to CD45 antigen and a monoclonal antibody that bindsto CD34 antigen, such that those cells that are AC133⁺5E12⁺CD45^(−CD)34⁻are selected.
 9. The method of any one of claim 1, 6, 7, or 8, furthercomprising the steps of: further enriching the population by selectingand eliminating from the population those cells that bind to monoclonalantibody 8G1.
 10. The method of any one of claim 1, 6, 7, 8, or 9,wherein all of the antibodies are fluorochrome conjugated.
 11. Themethod of any one of claim 1, 6, 7, 8, or 9, wherein all of theantibodies are conjugated to magnetic particles.
 12. The method of anyone of claim 1, 6, 7, 8, or 9, wherein the selecting is by flowcytometry.
 13. The method of any one of claim 1, 6, 7, 8, or 9, whereinthe selecting is by fluorescence activated cell sorting or high gradientmagnetic selection.
 14. The method of any one of claim 1, 6, 7, 8, or 9,wherein the population containing neural or neural-derived cells isobtained from any tissue which gives rise to neural tissue.
 15. Themethod of any one of claim 1, 6, 7, 8, or 9, wherein the populationcontaining neural or neural-derived cells is dissociated from any tissuewhich gives rise to neural tissue.
 16. A method for producing apopulation enriched for human central nervous system stem cells (CNS-SC)which can initiate neurospheres (NS-IC), comprising selecting from apopulation of neural or neural-derived cells for cells that bind tomonoclonal antibody AC133 or to monoclonal antibody 5E12 or to bothmonoclonal antibody AC133 and monoclonal antibody 5E12, and furthercomprising the step of further enriching the population by selecting andeliminating from the population those cells that are CD45⁺, CD34⁺, orCD45⁺CD34⁺, wherein the resulting population is enriched for NS-IC ascompared to the population of neural or neural-derived cells.
 17. Themethod of claim 16, further comprising the step of further enriching thepopulation by selecting and eliminating from the population those cellsthat bind to monoclonal antibody 8G1.
 18. A method for isolating aneurosphere initiating stem cell (NS-IC), comprising: a) combining apopulation comprising neural cells or neural-derived cells containing afraction of NS-ICs with monoclonal antibody AC133 or monoclonal antibody5E12 or both; b) selecting the cells that bind to monoclonal antibodyAC133 or to monoclonal antibody 5E12 or to both monoclonal antibodyAC133 and to monoclonal antibody 5E12, wherein the selected cells areenriched in the fraction of NS-ICs as compared with the population ofneural cells; c) combining said enriched fraction obtained in step b)with a monoclonal antibody that binds to CD45 antigen or a monoclonalantibody that binds to CD34 antigen or both; d) selecting andeliminating CD45⁺, CD34⁺, or CD45⁺CD34⁺ cells, wherein the remainingcells are further enriched in the fraction of NS-ICs as compared withthe enriched fraction obtained in step b); e) introducing at least onecell from the enriched fraction obtained in step d) to a culture mediumcapable of supporting the growth of NS-IC; and f) proliferating theintroduced cell in the culture medium.
 19. The method of claim 18,wherein the culture medium capable of supporting the growth of NS-ICcomprises a growth factor selected from the group consisting ofleukocyte inhibitory factor (LIF), epidermal growth factor (EGF), basicfibroblast growth factor (FGF-2) and combinations thereof.
 20. Themethod of claim 18, wherein the culture medium capable of supporting thegrowth of NS-IC further comprises a neural survival factor, NSF.
 21. Themethod of any one of claim 1, 6, 7, 8, 16, or 18, wherein the populationcontaining neural or neural-derived cells is obtained from a neurosphereculture or an adherent monolayer culture.